Geographic origin, ancestry, and death circumstances at the Cornaux/Les Sauges Iron Age bridge, Switzerland

Cornaux/Les Sauges (Switzerland, Late Iron Age) revealed remnants of a wooden bridge, artifacts, and human and animal skeletal remains. The relationship between the collapsed structure and the skeletal material, whether it indicates a potential accident or cultural practices, remains elusive. We evaluate the most plausible scenario for Cornaux based on osteological, taphonomic, isotopic, and paleogenomic analysis of the recovered individuals. The latter amount to at least 20 individuals, mostly adult males. Perimortem lesions include only blunt force traumas. Radiocarbon data fall between the 3rd and 1st c. BCE, although in some cases predating available dendrochronological estimates from the bridge. Isotopic data highlight five to eight nonlocals. No close genetic relatedness links the analyzed skeletons. Paleogenomic results, the first for Iron Age Switzerland, point to a genetic affinity with other Central and Western European Iron Age groups. The type of skeletal lesions supports an accidental event as the more plausible explanation. Radiocarbon data and the demographic structure of the sample may suggest a sequence of different events possibly including executions and/or sacrifices. Isotopic and paleogenomic data, while not favoring one scenario over the other, do support earlier interpretations of the last centuries BCE in Europe as a dynamic period from a biocultural perspective.

this bridge was built around 135 BCE and underwent partial repairs between 120 and 115 BCE, and possibly 105 BCE (this terminus ante quem is based on an ornate plate originally interpreted as a cart element, but which could be attributed to the bridge, according to Ramseyer 4 ).
The archaeological material fits well into this chronological spectrum.Typologically, the pottery (126 vessels, 75% of which was coarse ware) and the iron and bronze finds (weapons, ornaments, tools, utensils, a coin, etc.) can be dated to the end of the Second Iron Age, precisely to the La Tène D1 phase (150-90 BCE), with the exception of a few older objects (La Tène C) concentrated in upper part of the slope and attributed by H. Schwab to a "habitat" (Schwab 5 also reports some isolated Roman and Neolithic finds, which were well circumscribed in plan and stratigraphy and therefore do not call into question the homogeneity of the whole).
The site also yielded a large number of animal bones (cattle, horses, pigs, goats, sheep, a goose, an otter etc.) as well as human skeletons, both whole and partial, five of which presented the exceptional preservation of brain tissue or organic endocranial remains.Many of these skeletons were intermingled with the beams of the bridge deck and framework (Fig. 1).
The zooarchaeological study highlighted that the only animal specimens preserving the anatomical connection were the ones found on the lower bank and the bed of the river: cattle (MNI:3) and horses (MNI: 2).The other domestic species (e.g., pigs and sheep) were represented by fragmentary isolated bones in some case showing traces of cutting and butchering and were localized in the upper part of the slope and related to a possible settlement.
The suggested interpretation is that there were only horses and cattle involved in the possible convoy crossing the bridge 6,4 .The arrangement of the finds, the stratigraphy, and the taphonomy of the remains led H. Schwab to a catastrophic interpretation.She concluded that the bridge likely collapsed under the effect of a violent flood carrying a convoy including animals, people, and their goods into the water 5,7 .This interpretation, which guided H. Schwab from the beginning of the excavation, also relied on anthropological and archaeozoological analyses conducted by M.R. Sauter, G. Pilleri, and U. Imhof.It was immediately questioned by many authors 8,9,10,11 because of the indirect implications of this event-driven approach for the understanding of the neighboring (3 km upstream) eponymous site of La Tène 12 , which is partially similar in appearance 13,14 .
The recent archaeological re-examination by Denis Ramseyer 4 has generally confirmed the relevance of H. Schwab's interpretation according to sedimentary processes, which were also corroborated by geoarchaeologists Jean-Pierre Garcia & Christophe Petit 15 .However, it remains possible, or even probable, that before the collapse of the bridge around 100 BCE, this crossing point on the Thielle had already hosted "sacrificial" depositsas evidenced in particular by the isolated find of a bronze sheet of a sword scabbard methodically folded following documented practices of the ritual mutilation of weapons now firmly attested in the Celtic world in the 3 rd and 2 nd centuries BCE 16 .
The first morphological analysis of the skeletal remains from the site was provided by Sauter 17 and included a first estimate of the minimum number of individuals (20), demographic profiles (17 adults: 12 males, 2 females, and 3 indeterminate and 3 non adults) as well as data on the presence of traumatic lesions, stature, and anthropometric patterns.

Bone collagen extraction and mass spectrometric analyses (Carbon, Nitrogen, Sulfur stable isotopes)
Stable carbon (δ 13 C) and nitrogen (δ 15 N) isotope ratios in bone collagen are widely used to reconstruct ancient human diets 18 .δ 13 C provides insights about the dietary contribution of C3 and C4 plant products 19 .δ 15 N values reflect the trophic level of an organism, and the relative intake of animal and plant proteins.Sulfur isotope ratios (δ 34 S) are influenced by local factors, differentiate between terrestrial and marine environments, and help to assess marine contributions to the diet.δ 34 S values in freshwater ecosystems provide insights into dietary habits, and their correlation with local geology suggests their potential use in estimating territorial mobility 20,21 .
The extraction of bone collagen was performed following an acid-base-acid extraction method modified after Ambrose 22 23 , DeNiro 24 , and Longin 25 .After washing with distilled water, all samples were pulverized in a mix miller at 20 bps for 60 s.Then, 500 mg ± 3 mg of bone powder was demineralized with 10 ml of 1 M hydrochloric acid (HCl) for 20 min at room temperature.
The solution was washed until neutral (pH ~ 6-7).About 10 ml of 0.125 M of sodium hydroxide (NaOH) was added and left for incubation at room temperature for 20 h.The solution was then washed until neutral, and 10 ml of 0.001 M HCl was added.The samples were placed in a water bath for incubation at 90°C (10-17 h).The solubilized collagen was filtered (VitraPOR filterfunnel, porosity 16-40 μm) and lyophilized at 0.42 mbar for a minimum of 48 h.Of each sample, three times 3.0 mg ± 0.3 mg collagen was weighed into tin capsules.
The average of three measurements per sample was provided and was used for subsequent analyses.Results are reported in δ-notation in units of per mil (‰) according to the international standards of Vienna Pee Dee Belemnite (VPDB) for carbon, Ambient Inhalable Reservoir (AIR) for nitrogen, and Vienna Canyon Diablo Troilite (VCDT) for Sulfur.In addition, the laboratory internal standards STD R (collagen from cowhide from the EU project TRACE) and for most samples also STD BRA (collagen from Brazilian cowhide) were reported.Internal analytical errors were recorded as ±0.1‰ for δ 13 C, ±0.2‰ for δ 15 N, and ±0.3‰ for δ 34 S (standard error of the means calculated from 3 or 4 measurements).
We selected samples with a value of >1% collagen portion of dry bone (wt % = amount of extracted collagen/amount of bone powder used for extraction × 100).The molar C: N ratio ([%C/%N] × [14.007/12.011]) in the range of 2.9-3.6 was considered as a good quality collagen indicator 23 , same as when % C was in the range of 30%-47% and % N in the range of 11%-17.3% 22,23,26 .When at least one of the quality criteria was not within the stated range, we excluded the sample from further evaluation.We considered as good sulfur values when the C: N quality criteria were accepted, and in addition, for mammals and birds %S was within the range of 0.15%-0.35%,the C:S ratio between 300 and 900, and the N:S ratio between 100 and 300, while for archaeological fish %S within the range 0.40-0.85%, the C:S ratio between 175±50 and the N:S ratio between 60±20 27 .DIC samples were prepared for CO2 release as follows: a portion of sample was injected into 12-ml vials pre-filled with helium and 5 drops of 65% phosphoric acid and agitated in a Vortex mixed for 30 seconds.Afterwards, the vials were left at room temperature for 15 to 36 hours to reach a state of equilibrium 28 .The CO2 was separated from other residual gases by chromatography using a helium carrier gas in a Gas Bench (Thermo Finnigan, Bremen, Germany) system interfaced with a mass spectrometer 29 .Isotopic ratios in DIC samples were analyzed using a Delta XP mass spectrometer (IRMS).Three internal standards of Na2CO3 solution have been used (DIC-A, DIC-B and DIC-T), each with a different isotopic composition (-4.9‰, -9.50‰, +28.59‰ vs VPDB).These standards of about 15 L (preserved at room temperature by poisoning with mercuric chloride) were measured with an Elemental Analyzer online with a Delta Plus XL mass spectrometer (IRMS).Additionally, the carbonates were also measured with a Gas Bench (Thermo Finnigan, Bremen, Germany) system interfaced with a mass spectrometer (Delta XP).Precision calculated after correction of the mass spectrometer daily drift and from standards systematically interspersed in analytical batches was better than ± 0.1‰ for δ 13 C in DIC.

LARA Laboratory
The preparation of the bones followed Szidat et al. 30 and was slightly modified by the implementation of an ultrafiltration step, as recently realized in Steuri et al. 31 .The samples were cleaned by ultrasonication in ultra-pure water and ground to 0.5-1 mm with a ball mill.
The chemical treatment included the following steps: 0.5 mol/L hydrochloric acid (HCl) for 60 hr., 0.25 mol/L sodium hydroxide (NaOH) for 1 hr., 0.5 mol/L HCl for 1 hr, followed by a gelatinization in diluted HCl at pH 3 and 60°C overnight.The warm solution was filtered using precleaned Ezee-Filters, ultrafiltration was performed with Vivaspin™ 15 30 kDa molecular weight cut-offs (MWCO) ultrafilters (Sartorius) and the high-molecular-weight fraction was lyophilized.The extracted collagen was combusted and graphitized with an automated graphitization equipment (AGE).The 14 C measurements were performed with the accelerator mass spectrometry (AMS) system MICADAS using 14 C-free sodium acetate and the NIST standard oxalic acid II (SRM 4990C) for blank subtraction, standard normalization, and correction for isotope fractionations 32 .

Tandem Laboratory
The pre-treatment of bone samples followed a modified version of the protocol described in Longin 25 .The surface of bone samples was mechanically cleaned (scraping, in some cases sand blasting) and ultrasonically cleaned in boiled, distilled water (pH 3).The bones were then grinded in a mortar.0.8M HCl was added stirred (30 min, circa 10°C) (apatite removed).Distilled water kept at pH 3 was added to the insoluble fraction, which was heated while stirring (10 h, 90° C).The fraction to be 14 C-dated in the accelerator (early MICADAS machine from the Swiss company IONPLUS) was combusted to CO2 and graphitized using a Fe-catalyst reaction (see 33 for more details).

Oxygen stable isotope analysis: sample preparation and analysis
The analysis of oxygen isotopic ratios ( 18 O/ 16 O, δ 18 O) is a powerful tool for the reconstruction of past environments.These analyses are usually performed in animal and human tooth enamel with the aim of investigating paleoclimate and paleoseasonality, animal husbandry practices, but also human and animal territorial mobility and geographical provenance 34 .These analyses are also useful in the study of human sociocultural practices such as the process of breastfeeding, weaning, and past culinary practices (e.g., stewing and brewing) 35 .Oxygen isotopic signature in mammalian tissues reflects the isotopic values of the water ingested (as drinking water or contained in food) during the time of formation of the skeletal tissue.The ingested water often results to be closely in line with the isotopic composition of the local meteoric precipitation, which shows a strong geographical trend, influenced by different variables (e.g., latitude, distance from the coast, altitude, temperature, and humidity) 36,37 .
The δ 18 O ratios from dental enamel samples (from second or third molar or first or second premolar) of 10 individuals from the context of Cornaux, as well as from four animals (herbivores and pigs) (Supplementary Table S6), were measured.Samples weighing between 4.5 and 9 mg were chemically treated following protocols originally proposed by 38 , and modified by 39,40,41 .Enamel powder samples were treated for 4 h in 0.1 M acetic acid [CH3COOH] (0.1 ml solution/0.1 mg of sample), rinsed several times (at least five times) with distilled water and freeze-dried.Isotope measurements were performed on a Thermo Scientific Delta V Plus continuous flow-isotope ratio mass spectrometer (IRMS) equipped with a Gas-Bench II carbonate sample preparation and inlet system in the IsoTOPIK Laboratory at the University of Burgos Scientific and Technology Centre.Carbonate powder samples were digested in He-flushed borosilicate exetainers at 70 °C using water-free 99.9% phosphoric acid.
To assess the accuracy (external analytical precision) and to calibrate the obtained isotopic values, the results were measured against IAEA-603 (δ 13 C = +2.46‰,δ 18 O = -2.37‰)and NBS18 (δ 13 C = -5.014‰,δ 18 O = -23.2‰)international reference materials.Results are expressed as parts per thousand with respect to the Vienna Pee Dee Belemnite standard (‰ VPDB).The average 1 sigma internal precision error (ten injections per sample) is 0.03‰ for δ 18 O and 0.03‰ for δ 13 C.According to the reference material analyses, stable carbon and oxygen isotope values were measured accurately to the nearest 0.02‰ and 0.02‰, respectively.
Reproducibility was checked by duplicate analysis of some samples, and in the case of seemingly aberrant initial results, repeat sampling and analysis was undertaken.
We also analyzed δ 18 O from three water samples, two from the river Thielle and one from the close Lake Neuchâtel (in correspondence with the beach of La Tène) (Supplementary Table S6).The isotopic analyses of the water samples were carried out by injecting 1.8 microliters into a Picarro L-2140i.The replications of internal standards (contrasted with IAEA international standards) indicate errors of less than 0.1‰ and 0.5‰ for δ 18 O and δD, respectively.To approach geographical localization of the samples, we also converted carbonate δ 18 O values (VPDB) to the oxygen isotopic composition of drinking water (δ 18 ODW vs. VSMOW) using the equation provided by Chenery et al. 42 .
The Kernel density estimation and the calculation of the 95% density region of human oxygen values were calculated with the function hdr.den in the R package hdrcde 43 .The optimal bandwidth for the kernel density estimation was selected using the method of Sheather and Jones 44 , with function bw.SJ in R (version 4.3.2).

Strontium isotope analysis: sample preparation and analysis
Strontium isotopic analysis ( 87 Sr/ 86 Sr) of skeletal material is commonly used to detect geographic provenance and mobility among mammals, including humans 45,46 .The tooth enamel records the isotopic signal of when it was formed during the earliest stages of life, whereas the bone isotopic signal reflects a period closer to the time of death of the individual 47 Since the radiogenic isotope 87 Sr forms by radioactive decay from rubidium ( 87 Rb), the 87 Sr/ 86 Sr signature of a specific location is determined by the underlying bedrock age and its content of Rb 48 .A specific geological strontium signature is incorporated into the hard body tissues by direct substitution for calcium 49 since strontium enters the ecosystem without fractionation 50 .
The 87 Sr/ 86 Sr ratios from dental enamel samples (from second or third molar or first or second premolar) of ten individuals, as well as from four animals (herbivores and pigs) from Cornaux (Supplementary Table S5), four modern plants and four land snail shells from Rüfenacht (Bern) were measured.Sampling and the first preparation were carried out at the ANTARQBIO lab of the Universitat de València (Spain), while the chemical sample preparation and analysis were done in dedicated isotope facilities of the University of Cape Town (South Africa), as described below.Archaeological teeth, plants and snail shells were brought directly to the ANTARQBIO lab for their sampling and cleaning.The pretreatment of plant (all leaves) and land snail shell samples was realized following the procedures described in Copeland et al. 51 and Wong et al. 52 .The preparation included the cleaning, drying, incineration (at 500° C for 8 hours) and weighing (circa 20 mg of ash) for the plants and a first mechanic cleaning followed by a rinse with acetone and three more rinses with Milli-Q for 20 min each in the ultrasonic bath then the drying and weighing (⁓10-30 mg) for the snail shells.
The cleaned enamel samples (ca.20 mg for each type of material) were digested with 2mL bidistilled 65% HNO3 in a closed Teflon beaker placed on a hotplate at 140 °C for an hour.
Digested samples were then dried and redissolved in 1.5 mL of bi-distilled 2M HNO3.These re-dissolved samples were centrifuged at 4000 rpm for 20 minutes, and the supernatant was collected for strontium separation chemistry.A separate fraction for each sample in this step was used to calculate the concentration with 88 Sr intensity (V) regression equation built with SRM987 standard from NIST (National Institute of Standards and Technology, Gaithersburg, MD, USA).Strontium was then isolated with 200μl of Eichrom Sr.Spec resin loaded in Bio-Spin Disposable Chromatography Bio-Rad Columns following the method of Pin et al. 53 .The separated strontium fraction for each sample was dried down, dissolved in 2 ml 0.2% bi-distilled HNO3 and diluted to 200 ppb Sr concentrations for isotope analysis.87   Sr/ 86 Sr ratios were measured using a NuPlasma HR multicollector inductively-coupledplasma mass spectrometer (MC-ICP-MS).Sample analyses were referenced to bracketing analyses of SRM987, using a 87 Sr/ 86 Sr reference value of 0.710255 from NIST987.All strontium isotope data are corrected for isobaric rubidium interference at 87 amu using the measured signal for 85 Rb and the natural 85 Rb/ 87 Rb ratio.Instrumental mass fractionation was corrected using the measured 86 Sr/ 88 Sr ratio and the exponential law, and a true 86 Sr/ 88 Sr value of 0.1194 51 .Results for repeat analyses of an in-house carbonate standard NM95 ( 87 Sr/ 86 Sr = 0.708900; 2 sigma 0.000034; n=14) and an in-house ashed plant standard NAMISO316 ( 87 Sr/ 86 Sr = 0.720517; 2 sigma 0.000019; n=6) processed and measured with the batches of samples in this study are in agreement with long-term results for these two in-house standards ( 87 Sr/ 86 Sr; 0.708911; 2 sigma 0.000039; n=545) ( 87 Sr/ 86 Sr; 0.72051; 2 sigma 0.000043; n=8).
For every two batches one blank was added to assess the cleanness of the process; there was no peak and, thus, no contamination from external Sr in any of the batches.

Samples selection and sampling.
Based on the preservation and availability of the petrous portion of the temporal bone  S7).
Sampling was performed under clean conditions at the Laboratory of the Department of Physical Anthropology, Institute of Forensic Medicine, University of Bern, Switzerland.Before sampling, the surface of the bones was cleaned using a low-concentration H2O2 solution (3%) and then ddH2O.For the PP we generated bone powder from the petrous pyramid using a driller after removing the outer layer (approx.200 mg for each sample), and whole middle ear bones (69 -81 mg) were collected and stored for DNA extraction.
Further laboratory work was performed in a dedicated pre-PCR area of the aDNA laboratory at the Institute for Mummy Studies, Eurac Research, Bolzano, Italy, following all the strict rules required for aDNA analyses.

DNA Extraction
A silica membrane-based method was used for DNA extraction following a modified protocol based on 54,55 .The powder obtained from the sampling of the PP and the middle ear bones as a whole were dissolved in EDTA extraction buffer (0.5 M EDTA pH8, 20 mg/ml proteinase K) at 40° C overnight.After incubation, the extract was centrifuged at 5000 x g for 2 minutes to separate undissolved matter from the supernatant solution.The supernatant was then transferred into 4 ml Amicons (Ultra-4 centrifugal filter unit (30 kDa), Merck Millipore) and centrifuged at 2500 x g until reaching a final supernatant volume of 100 µL.The DNA concentrate was purified using the MinElute PCR Purification Kit (Qiagen) and eluted using EB buffer 56 .The purified DNA samples and the extraction blank control were quantified using the QuantiFluor® ONE dsDNA system (Promega 57 ) and stored at -20°C until library preparation.

Library Preparation
The extracted DNA was converted into double-stranded, double-indexed Illumina libraries for sequencing using a modified protocol according to Meyer & Kircher 58 .For library preparation, 25µl of DNA extracts were used and blank controls were included in every step, comprised of amplification.
Blunt-end repair of the DNA extract was carried out by combining the DNA extract with a 45 µl reaction mix, which consisted of NEBNext® End repair buffer (New England BioLabs Inc.), NEBNext® End repair enzyme mix (New England BioLabs Inc.), 10 mg/ml BSA (New England BioLabs Inc.), and ddH2O.The reaction mixture was incubated for 15 minutes at 25 °C, followed by 5 minutes at 12 °C.Subsequently, the reaction underwent purification using the MinElute PCR Purification Kit (Qiagen 56 ).For the adapter ligation step, a reaction was prepared containing 10X T4 DNA Ligase buffer, PEG 4000 (50%), Adapter Mix (5 µM each), T4 DNA Ligase (5 U/µl), and ddH2O.This reaction was incubated at 22° C for 20 minutes.After this step, another MinElute purification was performed 56 , and the elution volume was adjusted to 20 μl.To facilitate the fill-in of nicks on the unphosphorylated 3' ends of DNA fragments, the eluted samples (20 μl) were combined with a 20 μl reaction mix containing 10 X Thermopol reaction Buffer (New England BioLabs Inc.), dNTPs (2.5 µM each), and 4 U/μl Bst polymerase large fragment (New England BioLabs Inc.).The reaction was incubated for 20 minutes at 37 °C, followed by 20 minutes at 80 °C.For multiplexed sequencing, sample-specific indexing primers were added to each library through amplification.The amplification reactions had a total volume of 25 μl, with 3 μl of DNA library, NEBNext® Q5U ® Master Mix (New England BioLabs Inc.), BSA (New England BioLabs Inc.), and 10 µM of P5-primer and 5 µM of P7-primer.To increase library complexity and to reduce stacking, three amplification reactions with the same indexing primer were performed for each sample.The thermal profile started with 2 minutes at 95 °C, followed by 12 cycles with 30 seconds at 95 °C, 30 seconds at 58 °C, and 1 minute at 72 °C.The final extension step lasted for 10 minutes at 72 °C.After pooling the amplified libraries, MinElute purification was performed 56 , and the DNA was eluted in 25 μl EB-Buffer.Purified libraries were subsequently quantified using a High Sensitivity Bioanalyzer Kit (Agilent Technologies 59 ) and a QuantiFluor® ONE dsDNA System by Promega 57 .

Molecular Screening and In-Solution Target Enrichment
The indexed libraries were shotgun sequenced at the facility Macrogen (Seoul) on a HiseqX platform (150 bp PE) (Supplementary Table S8).All eleven samples fulfilled the quality criteria (presence of damage pattern of aDNA and content of human endogenous DNA ≥ 1%) and were then enriched for human DNA using the "Twist Ancient DNA"kit (Twist Bioscience) with a modified protocol optimized for aDNA 60 .The procedure was additionally adjusted by reducing the cycle numbers for post-enrichment amplification from 23 to 15.The reagent includes 1 434 155 probes targeting 1 352 535 SNPs of the human genome (including among others the core 1 240k SNPs, 81 925 on the Y-Chromosome, and 94 586 phenotypically relevant targets) 61 .For this study, the mitochondrial panel from Twist was spiked in during the experiment.
The library quantity input for the target enrichment ranged between 356.5 -1300 ng, to reach this quantity, adapted libraries were amplified several times and then pooled and dried at 30 °C using a vacuum concentrator (Eppendorf).For the hybridization, two different reactions were prepared: The probe solution contains the Twist Hybridization Mix, the Twist Custom Panel -AncientDNA_1.41MSNP, and the Twist Mitochondrial Panel, and the blocker solution is composed of the Twist Blocker Solution and the Twist Universal Blockers which are added to the dried indexed samples for resuspending.The probe solution is heated to 95°C for 2 minutes and then immediately cooled on ice for 5 minutes.Meanwhile, the blocker solution containing the resuspended indexed library pool is heated at 95°C for 5 minutes.Both the probe solution and resuspended indexed library pool are equilibrated at room temperature for 5 minutes.After mixing the two solutions, we added the Twist Hybridization Enhancer on top of the entire capture reaction.The hybridization reaction was incubated at 62°C for 16 hours in a thermal cycler and was then transferred to the Twist Streptavidin Binding Beads and Binding Buffer mix and incubated on a shaker at room temperature for 30 minutes in order to bind the targets to the beads.The mix of Streptavidin Binding beads and hybridization reaction was washed in several steps with the Twist Wash Buffer 1 (at room temperature) and Twist Wash Buffer 2 (at 48 °C), removing in between the supernatant by pelleting the beads on a magnetic particle collector, in order to remove the non-target DNA.After the last washing step and removal of the supernatant, ddH2O was added to the beads, and the Streptavidin Binding Bead slurry was incubated on ice until settled.Half of the Streptavidin Binding Bead slurry was used for postenrichment amplification using the Twist Equinox Library Amp Mix and the Twist Amplification Primers (10 uM) following initialization at 98°C for 45 seconds, then 15 cycles of 98°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds, followed by a final extension at 72°C for 1 minute.The amplified enriched libraries were then purified using the Twist DNA Purification Beads and 80% freshly prepared ethanol and then resuspended in EB buffer.Subsequent quantification of the enriched libraries, they were sequenced again on an Illumina Hiseq X (150 bp PE) system at the Macrogen Sequencing Centre (Seoul).

Bioinformatic analyses and aDNA authentication
A total of 703070376 (PE) new DNA sequences were generated for this study via shotgun sequencing and a total of 620278854 (PE) were generated via sequencing of enriched libraries.The sequencing reads were trimmed for removing adapters and merged following specific quality criteria: the number of overlapping bases of 11, and the minimum length of the assembled sequences of 25 62 .After merging the sequencing reads, alignment to the Human Reference Genome (GRCh37/hg19) and the revised Cambridge Reference Sequence (rCRS) was performed using BWA (v.0.7.17) setting the minimum mapping quality to 30 63 .With DeDup (v.0.12.8),reads with the same start and end and the same orientation (duplicates) were removed 64 .The ancient reads were examined for damage patterns, including fragmentation and misincorporation, using the software mapDamage (v.2.2.1) 65 .Exogenous human contamination was estimated at both nuclear and mitochondrial levels.For all samples, the software Schmutzi 66 was used to infer contamination levels on the mtDNA.For male individuals only, the method implemented in ANGSD (Analysis of Next Generation Sequencing Data) was applied to estimate contamination based on X-Chromosome data 67 .For this analysis, we excluded the pseudoautosomal region of the X chromosome and used a minimum base and mapping quality threshold of 30 with a coverage filter of 2. Thresholds for mtDNA contamination and nuclear contamination are ≤ 5% and ≤ 3% respectively.The results of both methods are reported in Supplementary Table S11.
For the two samples that show modern human contamination, we applied PMDtools with a quality threshold of 30.This software utilizes an approach that assesses aDNA damage patterns incorporating postmortem damage (PMD), base quality scores, and biological polymorphism in order to distinguish degraded DNA sequences that are unlikely to originate from modern contamination.This method assigns to each sequence a PMD score, with positive values indicating strong evidence that the sequence is indeed of ancient origin 68 .Data generated from shotgun sequencing and enrichment that met the set quality criteria, were merged for each individual, and merged data was used for downstream analyses in which BAM files after quality filter q30 were used.In Table S8 we summarize quality statistics for shotgun data, and in Supplementary Table S9 the ones from merged data.

Sex determination and unilinear transmitted markers
Biological sex was determined by calculating the ratio of sequences aligning to the X and Y Chromosomes as reported in 69 .We conducted a second verification using the method described in Mittnik et al. 70 .We applied these methods to all 10 samples since those filtered by PMD-tools retained a sufficient number of human reads requested by the two methods (100000 and 1000 respectively) for the accurate performance of the genetic sex determination (Supplementary Table S9).
To determine the mtDNA haplogroups, we used reads mapped to the reference mitochondrial genome (rCRS) from the consensus file generated by Schmutzi in 6 samples.For two samples we extracted a vcf file from the rescaled bam file generated by MapDamage.To ensure the accuracy of the haplogroup assignment, we established a threshold of a mean coverage of the mtDNA genome of 3 X (Supplementary Table S11).Since the two contaminated samples did not reach the set threshold after PMDtools filtering, we included in the results only eight samples from Cornaux.We exported the generated files and used them for the assignment in HaploGrep3, an automated mitochondrial haplogroup assignment tool available via https://haplogrep.i-med.ac.at/ (based on PhyloTree; mtDNA tree build 17, available via www.phylotree.org/) 71.
To identify the Y-Chromosomal haplogroups of male individuals, we used reads that mapped to the Y-Chromosome SNPs (human reference genome hg19) with a base quality ≥ 30.
Using the Yleaf software we assigned the Y-Chromosomal haplogroups 72 .To ensure accuracy, we verified any mutations found in the VCF files against the most recent phylogenetic tree of the human Y-Chromosome (ISOGG; Version: 15.73 Date: 11 July 2020).

Kinship analyses
For evaluating the genetic relatedness among individuals in Cornaux, we applied three different methods based on autosomal markers.The READ method (Relationship Estimation from Ancient DNA 73 and the TKGWV2 method (Thomas Kent Genome-Wide Variants 2 74 were used to infer relatedness up to the 2 nd degree.The KIN 75 method can infer genetic relationships up to 3 rd degree.
READ calculates and normalizes a mismatch rate across the genome to determine the degree of relationship between pairs of individuals.By employing genotype likelihoods and allele frequencies, READ estimates the probability of sharing zero, one, or two alleles that are identical by descent.This method requires a mean coverage of human reads of at least 0.1 X.
TKGWV2 can infer 1 st -and 2 nd -degree relatedness with as little as 0.026 X average coverage and uses genotype likelihoods and population allele frequencies of genome-wide variants present in the 1000 Genomes Project Phase 3. KIN, instead, uses a Hidden-Markov-Modelbased approach to identify biological kinship up to 3 rd -degree using at least 0.05 X sequence coverage.Additionally, it allows distinguishing between sibling and parent-child relationships.
To further verify and ascertain additional degrees of relatedness, we considered information from mtDNA haplotypes and Y-Chromosomal haplogroups, in conjunction with archaeological, chronological, and anthropological data.

Comparative Analyses and Dataset
For genome-wide downstream analyses, we used merged (shotgun + enrichment) data from eight non-related samples that cover a minimum of 50000 SNPs on the 1240k dataset.We genotyped the Cornaux samples at each SNP using samtools mpileup 76 , with a minimum base and mapping quality threshold of 30.To reconstruct pseudo-haploid genotypes, the pileupCaller tool was applied.This tool, available at https://github.com/stschiff/sequenceTools, randomly   Outliers for at least one isotopic system are highlighted in red.See also Table 1.

Figure S2 .
Figure S2.Plot (left) and output (right) of an Oxcal model including only the skeletons associated to wood beams analyzed dendrochronologically.The model considers two sequences, each one defined by a terminus post quem corresponding to the dendrochronological estimates for the beams.

Figure S3 .
Figure S3.Plot (left) and output (right) of an Oxcal model with all radiocarbon dates and a single phase constrained by the estimated chronological extremes of La Tène C2-D1 (ca.200-80 BCE).The boundaries consider an error of ±10 years

Figure S5 .
Figure S5.δ 13 C and δ 15 N values at Cornaux/Les Sauges.(a) Comparison of Cornaux with other late Iron Age Swiss contexts; (b) human and faunal isotopic values from Cornaux.Proxies of freshwater fish are pikes from Auvernier/La Saunerie (Lake Neuchâtel, Neolithic-Bronze Age).Red dots indicate individuals that resulted outliers for 87 Sr/ 86 Sr, δ 18 O and/or δ 34 S. The two isotopic outliers COR-3 and COR-10 are highlighted due to their highest δ 13 C values.

Figure S6 .
Figure S6.Overview of the misincorporation pattern at the end of the DNA fragments.The deamination damage pattern suggests the authenticity of the ancient DNA of all samples from Cornaux

Fig. S8 .
Fig. S8.Principal component analysis of ancient samples from Cornaux and other published ancient individuals from the Iron Age (IA) projected onto the genetic complexity of present-day populations of western Eurasia.Individuals from Cornaux are represented in red symbols whereas circles are used for present-day individuals and diamond-shaped symbols represent published ancient IA individuals from Europe.Different colors indicate the geographic origin.

Figure S9 .
Figure S9.Results of unsupervised clustering analysis (ADMIXTURE; K=7) of the Cornaux samples.On the left, ancient samples are shown that represent the different main ancestry components with different colors: the Western-Hunter-Gatherer component (WHG -here from Mesolithic individuals from central Europe -Switzerland and southern Europe -Italy) is represented in yellow.The Eastern Hunter-Gatherer Component (EHG -from Mesolithic individuals from Russia) is depicted by a combination of yellow, red, and blue colors.The Hunter-Gatherer component from Mesolithic individuals from the Caucasus and the Neolithic-related component, represented by Neolithic Individuals from Iran, are shown in purple.The green color displays the Neolithic-related component from Anatolia (here from Neolithic individuals from Turkey).Finally, the Steppe-related component (from Early Bronze Age -EBAindividuals from Russia; Yamnaya) is indicated by a mix of red-blue and purple colors.We also represent here published individuals from Neolithic and Bronze Age (BA) -Switzerland.The right side displays the Cornaux individuals that show a similar distribution of the different ancestry components, with minor differences (e.g.presence of the purple color).

Fig. S10 .
Fig. S10.Digitization of the original archaeological plan showing the spatial distribution of the remains and the range of analyses performed on each individual.The original plan indicates only the position of COR-21 and COR-23 with no drawing of the remains.

Fig. S11 .
Fig. S11.Cross-Validation (cv) plot with values for unsupervised clustering analyses by ADMIXTURE (K=2 to K=13).The lowest cv value indicates the K with the lowest error.

Fig. S12 .
Fig. S12.Results of unsupervised clustering analysis (ADMIXTURE; K=7) of all ancient samples used for this analysis.

Figure S13 .
Figure S13.Principal component analysis of ancient samples from Cornaux projected onto the genetic complexity of present-day populations of western Eurasia.Red symbols are used for ancient individuals from Cornaux whereas circles are used for present-day individuals.Different colors indicate the geographic origin.